In 1973, Bishop discovered the Rotavirus in Australia firstly. The rotavirus is a double stranded RNA virus and belongs to the Reoviridae family. The rotavirus causes acute gastroenteritis in infant and is infected via fecal-oral route after an incubation period of about 1 to 3 weeks. The disease is severe in 6 to 24 week infants, but is mild or asymptomatic in neonate or most of adults. Thus, the acute infectious diarrhea by the rotavirus is a main cause of the death in the world. Moreover, it is estimated that about a million of patients are died of the infectious diarrhea by the rotavirus in the developing countries [see reference: Blacklow, N. R. & Greenburg, H. B., (1991) Viral gastroentertitis N. Engl. J. Med., 325:152-164, 1991]. Therefore, world health organization (WHO) considered more efficient suppression and prevention for the infection by the rotavirus the first research subject [see reference: Glass, R. I et al., (1994) Rotavirus vaccines: success by reassortment Science 265-1389-1391].
The rotavirus is usually globular shape and is named after the outer and inner shells or double-shelled capsid structure of the same. The outer capsid is about 70 nm, and inner capsid is about 55 nm in diameter, respectively. The double-shelled capsid of the rotavirus surrounds the core including the inner protein shell and genome. The genome of the rotavirus consists of double stranded RNA segments encoding at least 11 rotavirus proteins. The inner capsid includes VP6 and VP2 proteins. VP4 and VP7 lie in outer side of the double-shelled capsid and constitute the outer capsid. Depending upon the antigenicity of VP6 which is a group-specific antigen, the rotavirus is divided into seven groups, A to G. VP2 protein is related to the synthesis of RNA. Group A rotavirus is further divided into the G-type (glycoprotein type) on the basis of the glycoprotein VP7, and P-type (protease-cleaved protein) on the basis of the VP4 which are associated with an important immunogenicity of the virus by forming a neutralizing antibody [see references: Estes M. K., et al., (1987) Synthesis and immunogenicity of the rotavirus major capsid antigen using a baculovirus expression system, J. Virol. 61:1488-1494; Estes M. K. & Cohen J., (1989) Rotavirus gene structure and function. Microbiol. Rev. 53:410—419; Desselberger U & McCrae M. A., (1994) The rotavirus genome. Curr. Microbiol. Immuno. 185:31-66].
The methods to treat the acute diarrhea disease caused by the rotavirus were restricted to administer non-specific physical strength supplements such as water and electrolyte. Thus, the vaccine as an effective therapeutic agent has been required for completely preventing the human from the all serum-type of human rotavirus. As an attempt, a live vaccine of attenuated human rotavirus, animal rotavirus such as cow, or reassortants comprised of RNA segments derived from different serotypes human and animal rotavirus have been used. As a result of such study, Wyeth Laboratories, a manufacturing company of vaccines, reported first rotavirus vaccine, RotaShield (trademark). The vaccine was produced by reassorting rotavirus of Rhesus monkey and human rotavirus of 3 serum types and was firstly approved by Food and Drug Administration (FDA) in the world in 1998. However, the FDA's approval to the vaccine was tentatively canceled in 1999, because of the side effects such as intussusception [MMWR Morb. Mortal Wkly. Rep., (1999) 5;48(43):1007].
Apart from the above, a study that the part of rotavirus is produced on a large scale through genetic recombinant technology and is used as a subunit vaccine is proceeding. In this point, many researchers have studied the expression of the rotavirus capsid proteins in E. coli expression system, baculrovirus expression system or mammalian expression system [Smith R. E. et al., (1989) Cloning and expression of the major inner capsid protein of SA-11 simian rotavirus in E. coli. Gene 79:239-248; Tosser G. et al., (1992) Expression of the major capsid protein VP6 of group C rotavirus and synthesis of chimeric single-shelled particles by using recombinant baculoviruses. J. Virol. 66:5825-5831; Ito H. et al., (1997) Expression of the major inner capsid protein, VP6, of avian rotavirus in mammalian cells. Vet. Microbiol. 49:257-265].
However, it is difficult to produce the rotavirus on a large scale by cell culture, because the rotavirus is infected through the mucus cell. That is, because it is difficult to produce the virus protein particles similar to natural virus in prokaryotic expression systems such as E. coli expression system, the particle does not elicit antigenecity. In view of contamination hazard during culture process, purification problem, high cost and low yield, the mammalian expression system is not satisfactory.
Also, most of disease caused by rotavirus is commonly occurred in developing countries which are deficient in sanitary facilities and vaccine supplement. Even if technical success of the method for producing the virus structural proteins in other cells and the vaccine composition comprising the proteins is possible, long time and many studies are required for commercializing the method in view of high production cost and sale price. On the other hand, if the rotavirus structural protein can be produced by using plant, especially edible plant and can be used for producing the vaccine, it is very economical in view of low production cost, no purification process, and efficiency in transport and storage.
Therefore, the present inventors found that rotavirus structural proteins capable of inducing the mucus and systemic immune response can be produced in the plant cell which is transformed with plant expression vector including rotavirus capsid gene, and then the plant cell is cultured under the suitable condition. Also, the vaccine composition comprising the rotavirus structural proteins can be obtained.
Other features and advantages of the invention will be apparent from the following detailed description, and from the examples.